PROJECT SUMMARY Human cells possess many intrinsic mechanisms that provide resistance to incoming virus infections, and a better understanding of these natural processes would be valuable in our ongoing fight against existing and emergent viral diseases. The interferon-induced transmembrane proteins (IFITMs) are cellular factors that potently block the fusion of multiple viruses. They are present in cells at steady state and also accumulate to higher, more effective levels during infection. IFITM3 in particular reduces the severity of influenza virus infections in both mice and humans. However, we currently lack a mechanistic understanding of how IFITM3 prevents influenza virus fusion. Likewise, we do not fully understand the regulatory processes controlling the abundance of IFITM3 in cells. We seek to bridge these fundamental gaps in our knowledge with two specific aims that will bring us closer to our long-term goal of designing IFITM3-based antivirals. Aim 1 will determine the molecular mechanism by which IFITM3 alters cellular membranes to prevent virus fusion. We have newly identified a short amphipathic helix within IFITM3 that we show is required for antiviral activity. Given that amphipathic helices are well characterized to induce membrane curvature, we will determine the ability of this helix to associate with and alter membranes, and will define its role in inhibiting influenza and other viruses. This work will provide the first evidence for an amphipathicity-based mechanism of action for the IFITMs. Aim 2 is based on our discoveries that the steady state level of IFITM3 in cells is conversely negatively regulated by the E3 ubiquitin ligase NEDD4 and positively regulated by the tumor suppressor PTEN. We have previously shown that NEDD4 directly ubiquitinates IFITM3, targeting it for degradation in lysosomes. We will now interrogate the mechanism by which PTEN promotes IFITM3 levels and resistance to influenza virus infection. We will examine which enzymatic activity of PTEN is involved in regulating IFITM3, we will determine whether PTEN and NEDD4 are involved in the same regulatory circuit, and finally, we will examine the involvement of PTEN in IFITM3-mediated resistance to infection in vivo using newly generated mouse models. Overall, these two independent aims will reveal complementary mechanisms that control IFITM3 activity and cellular abundance.